Cytochrome P450 monooxygenases (hereinafter “CYP450s”) form a large diverse gene family with about 246 isoforms in Arabidopsis and 372 identified in rice. CYP450s are hemoproteins that convert a broad range of substrates to more or less bioactive products. The reaction cycle catalyzed by CYP450s requires the sequential input of two reducing equivalents (i.e., two electrons and two protons). The reducing equivalents for the CYP450-catalyzed reaction are supplied by either NADPH or NADH, depending on the type of redox system concerned, and electron transfer is mediated by two co-factors, one of which is FAD; the other being either FMN or an iron-sulfur FeS2 redoxin (ferredoxin) or, in the microsomal system, cytochrome b5. In particular, the majority of plant CYP450s utilize an electron transport chain which consists of an FAD-containing NADPH-dependent oxidoreductase (Werck-Reichhart, Trends in plant science 5 (2000) 116-123). The mitochondrial system in mammalia bears many similarities with the plant P450 electron transport chain and both systems are generally referred to as Class I (see Lewis and Hlavica, Biochimica et Biophysica Acta 1460 (2000) 353-374, as well as references contained therein). CYP450s are critical in numerous plant metabolic pathways, including biosynthesis of hormones, secondary metabolites and lipids, particularly lignin and pigment biosynthesis, detoxification of harmful compounds, and are considered important in the evolution of land plants. Inhibitors of CYP450 activity include 1-aminobenzo-triazole, tetcyclacis, piperonyl butoxide, cinnamonic acid, and tridiphane.
Several approaches can lead to herbicide tolerant plants: a) modification of the target molecule of the herbicide, b) metabolic approach, i.e. making the compound non-hazardous. For the metabolic solution, one or more enzymes are needed, that catalyze the conversion of the herbicide to a non toxic compound. One source of such enzymes can be microorganisms isolated from nature. Bacteria, especially those of the order Actinomycetales, are known for their potential to detoxify soil by metabolizing xenobiotics, including herbicides (Cork et al, 1991; Schrijver et al., 1999; Caracciolo et al., 2010). These detoxifying reactions can be catalyzed by O-demethylases from Pseudomonas maltophilia DI-6, like it was shown for the herbicide Dicamba (Chakraborty et al., 2005; Wang et al., 1997).
In many other cases those reactions are catalyzed by CYP450's. Those can be plant derived (Pan et al., 2006), from algal (Thies et al., 1996) or microbial origin (O'Keefe et al., 1991). Among bacteria, especially actinomycetes offer a broad spectrum of CYP450's. The genome analysis of Streptomyes coelicolor revealed 18 CYP450's (Lamb et al., 2002), the Streptomyes avermitilis genome revealed 33 (Lamb et al., 2003). According to Nelson (2011), actinobacteria hold the largest number of CYP450's per genome.
Current enzymes available for metabolizing herbicides, such as, for example, saflufenacil, e.g. those described in WO2010/143743 particularly when expressed in plants, do not have particularly high activity. Thus, there is the need for the identification of further enzymes which can be used to metabolize herbicides.
The present inventors have characterized an Alopecurus species utilizing a mechanism of metabolizing herbicides. Furthermore, the inventors have isolated and characterized the novel herbicidemetabolizing CYP450 monooxygenases from this grass species.